A programmable CRISPR/Cas9-based phage defense system for Escherichia coli BL21(DE3)

Li Liu,Muzi Hu,Bin Xiong,Lijun Ye,Xueli Zhang,Changhao Bi,Tao Zhan,Dongdong Zhao

doi:10.1186/s12934-020-01393-2

Abstract

Escherichia coli BL21 is arguably the most popular host for industrial production of proteins, and industrial fermentations are often plagued by phage infections. The CRISPR/Cas system is guided by a gRNA to cleave a specific DNA cassette, which can be developed into a highly efficient programable phage defense system. In this work, we constructed a CRISPR/Cas system targeting multiple positions on the genome of T7 phage and found that the system increased the BL21’s defense ability against phage infection. Furthermore, the targeted loci on phage genome played a critical role. For better control of expression of CRISPR/Cas9, various modes were tested, and the OD of the optimized strain BL21(pT7cas9, pT7-3gRNA, prfp) after 4 h of phage infection was significantly improved, reaching 2.0, which was similar to the control culture without phage infection. Although at later time points, the defensive ability of CRISPR/Cas9 systems were not as obvious as that at early time points. The viable cell count of the engineered strain in the presence of phage was only one order of magnitude lower than that of the strain with no infection, which further demonstrated the effectiveness of the CRISPR/Cas9 phage defense system. Finally, the engineered BL21 strain under phage attack expressed RFP protein at about 60% of the un-infected control, which was significantly higher than the parent BL21. In this work, we successfully constructed a programable CRISPR/Cas9 system to increase the ability of E. coli BL21’s to defend against phage infection, and created a resistant protein expression host. This work provides a simple and feasible strategy for protecting industrial E. coli strains against phage infection.

Highlights

Escherichia coli is a major host for laboratory and industrial production of proteins, among which the BL21 series of strains are the most popular because they can expresses heterologous proteins at high levels [1]
Expression of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 with gRNA targeting the T7 phage genome increased the phage defense ability of E. coli BL21(DE3) To determine whether programed CRISPR/Cas9 can increase the ability of BL21 to defend itself against bacteriophages, we designed three different N20s, respectively targeting the genes of the tail tubular protein gp12, capsid assembly protein and 3.8 protein in the T7 phage genome (Fig. 1)
At MOI 0.02, the results showed that the OD600 of BL21(pcas9, pTgRNA) and BL21(pcas9, p3gRNA) was significantly higher than that of BL21(pCas9) with phage infection (Fig. 2)

Summary

Introduction

Escherichia coli is a major host for laboratory and industrial production of proteins, among which the BL21 series of strains are the most popular because they can expresses heterologous proteins at high levels [1]. BL21 can be easy infected by bacteriophages which can lead to enormous economic losses in industrial production [2], and there are no ideal solutions for this problem to date [2]. Wild type E. coli MG1655 strains have a CRISPR-Cas system, which belongs to type I CRISPR-Cas, comprised of CRISPR, Cascades (CRISPR associated complex for antiviral defense proteins) and Cas3 [10]. The complete CRISPR-Cas system can target different sites on the genomes of E. coli phages such as λ, T7, or T4, and lead to a delay in the growth of phage progeny in the infected culture [11]. E. coli BL21 does not carry any CRISPR-Cas system according to its genomic sequence, and according to predictions done using the CRISPRs web server (http://crispr.i2bc.paris-saclay.fr/?tdsourcetag=s_pcqq_aiomsg), suggesting that the sensitivity of BL21 to phages might due to its lack of adaptive immunity

Methods

Results

Conclusion